This invention relates to nucleic acid and amino acid sequences of a human S-adenosyl-L-methyltransferase and to the use of these sequences in the diagnosis, prevention, and treatment of neoplastic, immunological, and vesicle trafficking disorders.
Covalent modification of cellular substrates with methyl groups has been implicated in the pathology of cancer and other diseases (Gloria, L. et al. (1996) Cancer 78:2300-2306). Cytosine hypermethylation of eukaryotic DNA prevents transcriptional activation (Turker, M. S. and Bestor, T. H. (1997) Mutat. Res. 386:119-130). N6-methyladenosine is found at internal positions of mRNA in higher eukaryotes (Bokar, J. A. et al. (1994) J. Biol. Chem. 269:17697-17704). Hypermethylated viral DNA is transcribed at higher rates than hypo- or hemimethylated DNA in infected cells (Willis, D. B. et al. (1989) Cell. Biophys. 15:97-111).
Many pathways of small molecule degradation, such as those of neurotransmitters, require methyltransferase activity (Kagan, R. M. and Clarke, S. (1994) Arch. Biochem. Biophys. 310:417-427). Degradation of catecholamines (epinephrine or norepinephrine) requires phenylethanolamine N-methyltransferase. Hydroxyindole methyltransferase converts N-acetyl-5-hydroxytryptamine to melatonin in the pineal gland.
S-adenosylmethionine (AdoMet) is an important source of methyl groups for methylation reactions in the cell (Bottiglieri, T. and Hyland, K. (1994) Acta Neurol. Scand. Suppl. 154:19-26). Methyltransferase activity catalyzes the transfer of methyl groups from AdoMet to acceptor molecules such as phosphotidylethanolamine or the polynucleotide 5xe2x80x2 cap of viral mRNA (Montgomery, J. A. et al. (1982) J. Med. Chem. 25:626-629).
Members of the S-adenosylmethionine methyltransferase family (AdoMet-MT), utilize AdoMet as a substrate or product and harbor three common consensus sequence motifs. Motifs I and II are characteristically spaced between 34 and 90 (mode 52, median 52-54) amino acid residues apart; motifs II and III are spaced between 12 and 38 (mode 22, median 20-22) residues apart. Motif I comprises part of the AdoMet binding pocket; motif II may also be involved in binding AdoMet; the role of motif III is uncertain (Kagan, R. M. and Clarke, S. (supra)).
Messenger RNA N6-adenosine methyltransferase holoenzyme has been partially purified from HeLa cell nuclear extract to yield three subunits, an 875 kDa ssDNA-agarose binding protein, a 70 kDa AdoMet-binding protein, and an approximately 30 k-Da component with unknown function. The three components are absolutely required for RNA m6A-methylation activity (Bokar, J. A. (supra)).
The nematode Caenorhabditis elegans employs many of the same methyltransferase activities found in higher animals (Kagan, R. M. and Clarke, S. (1995) Biochemistry, 34:10794-10806). A C. elegans C27F2 gene product identified as a member of the methyltransferase family has now been described (Wilson, R. et al. (1994) Nature 368:32-38).
In their roles as a rate-limiting step in methyltransferase reactions, AdoMet-MTs have been identified as a target for psychiatric, antiviral, anticancer and anti-inflammatory drug design (Bottiglieri, T. and Hyland, K. (supra); Gloria, L. et al. (supra)). Sequence-specific methylation inhibits the activity of the Epstein-Barr virus LMP 1 and BCR2 enhancer-promoter regions (Minarovits, J. et al. (1994) Virology 200:661-667). 2xe2x80x2-5xe2x80x2-linked oligo(adenylic acid) nucleoside analogues synthesized by interferon-treated mouse L cells act as antiviral agents (Goswami, B. B. et al, (1982) J. Biol. Chem. 257:6867-6870). Adenine analogue inhibitors of AdoMet-MT decreased nucleic acid methylation and proliferation of leukemia L1210 cells (Kramer, D. L. et al. (1990) Cancer Res. 50:3838-3842).
The discovery of a new human S-adenosyl-L-methionine methyltransferase and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of neoplastic, immunological, and vesicle trafficking disorders.
The invention features a substantially purified polypeptide, human S-adenosyl-L-methionine methyltransferase (SAM-MT), having the amino acid sequence shown in SEQ ID NO:1, or fragments thereof.
The invention further provides an isolated and substantially purified polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or fragments thereof and a composition comprising said polynucleotide sequence. The invention also provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the amino acid sequence SEQ ID NO:1, or fragments of said polynucleotide sequence. The invention further provides a polynucleotide sequence comprising the complement of the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, or fragments or variants of said polynucleotide sequence.
The invention also provides an isolated and purified sequence comprising SEQ ID NO:2 or variants thereof. In addition, the invention provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:2. The invention also provides a polynucleotide sequence comprising the complement of SEQ ID NO:2, or fragments or variants thereof.
The present invention further provides an expression vector containing at least a fragment of any of the claimed polynucleotide sequences. In yet another aspect, the expression vector containing the polynucleotide sequence is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof, the method comprising the steps of: a) culturing the host cell containing an expression vector containing at least a fragment of the polynucleotide sequence encoding SAM-MT under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified SAM-MT having the amino acid sequence of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention also provides a purified antagonist of the polypeptide of SEQ ID NO:1. In one aspect the invention provides a purified antibody which binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:1.
Still further, the invention provides a purified agonist of the polypeptide of SEQ ID NO:1.
The invention also provides a method for treating or preventing a neoplastic disorder comprising administering to a subject in need of such treatment an effective amount of a purified antagonist to SAM-MT.
The invention also provides a method for treating or preventing an immunological disorder comprising administering to a subject in need of such treatment an effective amount of a purified antagonist to SAM-MT.
The invention also provides a method for treating or preventing a vesicle trafficking disorder comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising purified SAM-MT.
The invention also provides a method for detecting a polynucleotide which encodes SAM-MT in a biological sample comprising the steps of: a) hybridizing the complement of the polynucleotide sequence which encodes SEQ ID NO:1 to nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding SAM-MT in the biological sample. In one aspect the nucleic acid material of the biological sample is amplified by the polymerase chain reaction prior to hybridization.